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From 2012.igem.org

Labbook - TAL vector

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08/08/12

1.) MutPCR for pSB1C3 vector backbone in order to remove restriction sites vor BsmBI
2.) Transformation of Sirt2 and HAT5
3.) GGC in order to produce our MammoBrick

1.) MutPCR:

	Tube 1 [µl]	Tube 2 [µl]
Primer MutPCR fo [10 mM]	1	0,25
Primer MutPCR re [10 mM]	1	0,25
dNTPs [10 mM]	1	1
Phusion Buffer HF	1,25	1,25
Phusion Polymerase	0,25	0,25
DMSO	1	1
MgCl2	1	1
Template	1	1
ddH2O	5	6,5

Program for Thermocycler (20 cycles)

1.	95°C	5min
2.	95°C	50s
3.	80°C	50s
4.	72°C	3min
5.	72°C	7min
6.	4°C	//

2.) Transformation of Sirt2 and HAT5 in DH10B starin of E. coli using our standard protocol for transformation. Kanamycin resistance was used

3.) GGC in order to produce our Mammobrick:

Program for Thermocycler (20 cycles)

Contens	[µl]
BsaI [10 U/µl]	1,5
NEB Buffer 4	95°C	50s
ATP [10 mM]	1
BSA	1
TALEN fragment [40 ng]	2
exCMV promotor [20 ng]	0,5
PostORF [20 ng]	1,25
PuroORF [20 ng]	0,5
T7 Ligase	0,25
DTT [10 mM]	1
ddH2O	1

Program for Thermocycler (15 cycles)
1.      37°C      5min
2.      20°C      5min